When administered to rat pituitary cells grown in monolayer culture, progesterone (P) modulates biphasically gonadotropin secretion stimulated by GnRH or other secretagogues P enhances LH/FSH release by 3h and 12-24h later inhibits LH but not FSH secretion. P actions depend on estrogen pretreatment and are elicited by 10-7-10-10M P, concentration in the physiologic range in blood. Similar P actions are seen in vivo and are thought to determine LH/FSH release during the female reproductive cycle as well as following progestin-containing contraceptive treatments. My research's objective is to understand the intracellular mechanisms mediating gonadal steroid control of hypophysial function. This proposal will study P's actions using cell cultures highly enriched with gonadotrophs. Studies will explore the intracellular processing of P and investigate P actions on estradiol processing. Nuclear progestin+receptor complexes will be monitored quantitatively following exposure to 3H-P plus and minus the antiprogestin RU38486 to demonstrate the importance of the receptors for P action. The steroidal make-up of the 3H-progestin+receptor complexes will be analyzed chromatographically; the importance of 5Alpha-reduction for P action will be tested directly with a 5Alpha-reductase inhibitor. Similar approaches will examine if P suppression of estrogen processing can account for P's long-term inhibition of GnRH sensitivity. Other experiments will examine whether GnRH influences 32P04 incorporation into cytosolic, membrane or nuclear proteins and whether this mechanism(s) is important for gonadotropin release. SDS-PAGE procedures will be used to separate the labeled proteins; 32P04 incorporation will be quantified by densitometric analysis of the gel autoradiograms. These protein profiles will be compared to those obtained after cyclic nucleotide, A23187 or phosphatidic acid treatments to describe potential intracellular hierarchies mediating GnRH action. P influences on secretagogue-stimulated protein phosphorylation will also be monitored in an attempt to identify potential sites for GnRH-P synergy. Final studies will examine P's actions on the bioactivity/immunoactivity ratios of LH secreted by or retained in cells following GnRH and will determine if the changes can be attributed to variations in molecular species of LH. Similar studies will be run in vivo. The results of these studies will increase our understanding of steroid and neurohormone action in the adenohypophysis as well as provide information about the molecular mechanisms involved in gonadotropin release.